Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy

Abstract

Progress in adoptive T-cell therapy for cancer and infectious diseases is hampered by the lack of readily available, antigen-specific, human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes, but the therapeutic potential of human pluripotent stem cell-derived lymphoid cells generated to date remains uncertain. Here we combine induced pluripotent stem cell (iPSC) and chimeric antigen receptor (CAR) technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture. These iPSC-derived, CAR-expressing T cells display a phenotype resembling that of innate γδ T cells. Similar to CAR-transduced, peripheral blood γδ T cells, the iPSC-derived T cells potently inhibit tumor growth in a xenograft model. This approach of generating therapeutic human T cells 'in the dish' may be useful for cancer immunotherapy and other medical applications.

Figure 1

Differentiation of 1928z CAR–engineered T-iPSCs into CD19-specific functional T lymphocytes. (a) The study concept. Peripheral blood lymphocytes are reprogrammed to pluripotency by transduction with retroviruses encoding c-MYC, SOX2, KLF4 and OCT-4 (ref. 7). The resulting T-iPSCs are genetically engineered to express a CAR and are then differentiated into T cells that express both the CAR and an endogenous TCR. (b) In vitro T-lymphoid differentiation protocol. T-iPSCs were stably transduced with a bicistronic lentiviral vector encoding the 19–28z CAR and the fluorescent marker mCherry. mCherry⁺ CAR⁺ T-iPSCs are differentiated in three steps: (i) mesoderm formation (days 1–4), (ii) hematopoietic specification and expansion (days 5–10) and (iii) T-lymphoid commitment (days 10–30). Fluorescence microscopy images (below) show mCherry expression was maintained throughout the differentiation process. Scale bars,100 μM. (c) Flow cytometric analysis of 1928z-T-iPSC–derived cells at day 30 of differentiation. Representative plots are of at least five independent differentiations. (d) 1928z-T-iPSC-T cells were seeded into cultures of 3T3 cells or 3T3 cells expressing CD19 (3T3-CD19). Co-cultures shown 24 h after T-cell seeding; formation of T-cell clusters and elimination of the 3T3-CD19 monolayer are visible. Scale bars, 100 mM. (e) Flow cytometric analysis of CD25 and CD69 expression on the surface of 1928z-T-iPSC-T cells 48 h after exposure to 3T3 or 3T3-CD19 cells. (f) Luminex multiplex cytokine analysis of culture supernatant 24 h after seeding of 1928z-T-iPSC-T cells on 3T3 or 3T3-CD19 cells. Data are presented as mean of two independent experiments ± s.d.

Figure 2

Phenotypic profiling of 1928z-T-iPSC-T cells before and after CD19-induced expansion. (a) Unsupervised hierarchical clustering of 35 total transcriptomes, generated by an mRNA gene expression microarray, from 1928z-TiPCS-T cells at days 30–35 of differentiation (1928z-T-iPSC-T) and other human lymphoid cell subsets isolated for this study [CD3⁺TCRγδ⁺ cells (γδ-T), CD3⁺CD56⁺ cells, CD8⁺ cells and CD4+ cells] or downloaded from the NCBI repository GEO database (naive B cells, TCRVγ9 γδ T-cells before activation (γδ-T GEO) and after activation with BrHPP/IL-2 (bromohydrin pyrophosphate/interleukin-2) for 6 h (γδ-T 6h activ) or 7 days (γδ-T 7d activ) and resting NK cells). (b) Heatmap comparing the expression of indicated mRNA transcripts expressed in lymphoid and/or NK cells. Transcripts are classified according to known function and expression patterns. (c) Intracellular expression of the transcription factor PLZF (red histogram), compared to isotype control (black histogram), and surface expression of CD161 and CD3 in 1928z-T-iPSC-T cells, as assessed by flow cytometry. (d) Expansion of 1928z-T-iPSC-T cells after weekly stimulations with 3T3-CD19 cells in the presence of IL-7 (10 ng/ml) and IL-15 (10 ng/ml) for 4 weeks. Absolute cell numbers are shown. Arrows indicate restimulations with freshly irradiated 3T3-CD19 AAPCs. (e) Flow cytometric analysis of cell surface molecules and cytotoxic receptors in gated CD3⁺ 1928z-T-iPSC-T cells before and 7 d after expansion on 3T3-CD19 AAPCs. (f, g) qRT-PCR analysis of the expression of the indicated mRNA transcripts in 1928z-T-iPSC-T cells before and 7 d after expansion on 3T3-CD19 AAPCs. Data were normalized to the values of endogenous GAPDH and pre-expansion expression levels were used as reference. Graphs represent average of intra-assay technical triplicates. Error bars, mean ± s.d.

Figure 3

1928z-T-iPSC-T cells lyse CD19-positive tumor cells in vitro and in vivo. (a) In vitro 51Cr release assay of 7 d-expanded 1928z-T-iPSC-T cells (effectors) and the murine lymphoma cell line EL-4 expressing ovalbumin (EL4-OVA) or human CD19 (EL4-CD19) (targets). E/T, effector/target ratio. Representative of two independent experiments. (b) Flow cytometric analysis of 1928z-T-iPSC-T cells and syngeneic 1928z-transduced γδ (1928z-γδ) and αβ (1928z-αβ) T cells before their injection into tumor-bearing mice. Bottom: black histogram, untransduced cells; red histogram, transduced cells. Representative plots of two independent experiments. (c) NOD-SCID IL2Rγcnull mice were inoculated intraperitoneally with CD19+ Raji human Burkitt lymphoma cell line expressing a green fluorescent protein–firefly luciferase fusion protein (GFP/Luc). Four days later, T cells (4 × 105) described in b were injected intraperitoneally. No treatment indicates mice that were injected with tumor cells but not T cells. Tumor burden was measured biweekly by bioluminescent imaging. Images of representative time points are shown. Images of three mice from each group were intentionally selected to show mice relapsing after treatment. Disappearance of a mouse from the sequence of images indicates death of that mouse. (d) Kaplan-Meier curve representing the percent survival of the experimental groups described in b (1928z-T-iPSC-T: n = 4, 1928z-γδ: n = 5, 1928z-αβ: n = 7, no treatment: n = 6). Color-coded arrows depict death events not related to tumor growth in the corresponding groups. Statistical analysis between the treated experimental and the untreated control group, depicted here, was done using the log-rank test and P < 0.05 was considered significant.